The means by which a warm blooded animal, including a human, overcomes microbial pathogenesis is a complex process. Immunity to microbial pathogenesis is one means by which a warm blooded animal avoids pathogenesis, or suffers a less intense pathogenic state. Incomplete immunity to a given pathogen results in morbidity and mortality in a population exposed to a pathogen. It is generally agreed that vaccines based on live but attenuated micro-organisms (live attenuated vaccines) induce a highly effective type of immune response. Such vaccines have the advantage that, once the animal host has been vaccinated, entry of the microbial pathogen into the host induces an accelerated recall of earlier, cell-mediated or humoral immunity which is able to control the further growth of the organism before the infection can assume clinically significant proportions. Vaccines based on a killed pathogen (killed vaccine) are generally conceded to be unable to achieve this type of response. However, vaccines that contain a live pathogen present, depending on the level of attenuation, the danger that the vaccinated host upon vaccination may contract the disease against which protection is being sought. Therefore, it would be desirable to have a vaccine that possesses the immunising attributes of a live micro-organism but that is not capable of causing undesirable side effects upon vaccination.
However, it is important to note that the effective use of an attenuated bacterial strain as a vaccine candidate cannot be predicted merely by such level of attenuation. In this regard, the general approach for attenuating bacteria is the removal of one or more virulence factors (genetic modified organisms—GMOs), in most cases, however, virulence factors also play a role in inducing immunity as protective epitopes. In those cases, deletion of virulence factors unavoidably impairs the immunogenic capacities of the bacterium. This is of course an unwanted situation. Therefore, a live vaccine should preferably retain the antigenic complement of the wild type strain.
Moreover, once attenuation level is established, the immune response to a particular type of vaccine candidate and the success of a vaccine composition including such micro-organisms may still be influenced by many factors as detailed below:                a. The live attenuated vaccine strain should preferably have substantially no probability for reverting to its original state (usually a virulent wild type strain) and none of the genes manipulated should be complemented by other genes causing the bacteria to be capable of causing disease (stable mutations are preferred).        b. The presence of endotoxins in a live vaccine can be a disadvantage if not considered as these molecules can cause serious systemic reactions. Also, the administration of whole-cell vaccines is a classical risk factor for local reactogenicity (severe pain, local swelling and edema, panniculitis or ulcer, etc).        c. The viability and fitness of the attenuated GMO should not be drastically affected, as some replication is expected to occur in the body to create enough of the micro-organism and its antigens to stimulate the immune system. In fact, any mutation in a gene may interfere with replication or may damage the live micro-organism in the vial, causing the vaccine to be ineffective. Therefore, each type of genetic modification must be carefully evaluated for unexpected effects on the cell.        d. Moreover, gene sharing or protein moonlighting—a phenomenon by which a protein can perform more than one function—should be considered when selecting a gene target for genetic manipulation. Many proteins that moonlight are enzymes. One example is Glutamate racemase (Mud) which is a critical enzyme in cell wall biosynthesis but also plays a role in gyrase inhibition. Owing to its multifunctional character, the usefulness of MurI-targeted strategies cannot be predicted unless mutations in these genes are obtained and evaluated for the impact on the bacterial cell physiology.        e. In addition, the type of immune response elicited by a vaccine may not be appropriate to provide an adequate protection against infection (vaccine failure). The specific requirements for an effective vaccine will vary according to the nature of the pathogen. In the case of extracellular pathogens, the major antibodies provide adaptative mechanisms for the defense of the organism, while the presence of T cells is essential in controlling intracellular organisms. In consequence, live attenuated vaccines serve as better immunogens that killed bacteria or subunit compositions by means of simple multiplication, as well as by the modifications of bacterial antigens that occur during in vivo infection. Thereby, a live attenuated strain could engender a broader and adequate immune response, especially in the intracellular phase. In this sense, gene-targeted strategies of attenuation should be carefully tested in vaccine candidates, as the ability of the manipulated bacteria to exploit the natural pathways of infection could be potentially impaired and not trigger a broadly protective immune response.        f. In addition, irrespectively of the attenuation level or the type of immune response elicited, the number of doses administrated to achieve an acceptable level of protection with a specific GMO (effective and lasting) can be unsustainable for a vaccine schedule.        g. Furthermore, the route of administration of a vaccine can determine the type of immune response mounted and to be crucial for its success. Depending on the route of administration, the vaccine may enter the organism in different ways: skin (in this case the antigen is taken up by Langerhans cells that act as antigen-presenting cells in the T-zone of regional lymph nodes); mucosa (here the capture of antigen is carried out mainly by M cells and the immune response is developed in the Peyer's patches) or blood (the antigen would target the spleen where it would be processed by splenic macrophages). Consequently, once attenuation level is established for a GMO, the site of vaccine administration could determine the failure or success of vaccination. In this regard, it has been demonstrated that intramammary but not intraperitoneally administration of a live attenuated S. aureus strain significantly decreases the bacterial load in mammary glands after challenge with the wild type strain. The proposed vaccine candidate, S. aureus 8325-4 A523, is a temperature-sensitive mutant isolated after mutagenesis with nitrosoguanidine, which replicates well at low temperatures (below 32° C.) but undergoes a limited number of divisions when transferred to the mammalian body temperature. The authors performed challenge experiments with the S. aureus 8325-4 wild type strain to compare bacterial loads in mammary glands between vaccinated and non-vaccinated animals as measure of vaccine protection efficacy. These authors concluded the following: “The number of S. aureus CFU recovered from the mammary glands of mice immunized by the intramammary route was significantly lower (7×102 CFU) than that found in control mice (1.5×105 CFU). Conversely, the number of CFU recovered from mammary glands of mice immunized by any of the intraperitoneal protocols was as high as that recovered form control mice glands (P>0.5)”. Therefore, even with a potentially good candidate, the route of administration can determine the efficacy of the live attenuated mutant as a vaccine.        h. Lastly, a vaccine can be unable to induce cross-reactive antibodies against multiple strains of the same bacterial species. Although live attenuated strains can elicit antibodies that are protective in animal models, this protection is generally seen only when the parental strain used to create the vaccine strain is also used in the challenge studies. Broad-based protection against other strains usually is not reliable generated or tested. Moreover, antibodies produced, although adequately elicited and cross-reactive, may not last long nor be protective in a model of challenge with the wild type pathogen.        
In summary, a live vaccine should be sufficiently attenuated (or a-virulent) to avoid unacceptable pathological effects, but on the other hand it must elicit an adequate immune response capable of conferring a lasting protection in the host against the disease (protective immunity) independently of the bacterial strain.
Demonstrating that a live vaccine is sufficiently attenuated (or a-virulent) to avoid unacceptable pathological effects and elicits an adequate immune response capable of conferring a lasting protection in the host against the disease (protective immunity) independently of the bacterial strain, is not an easy task. In this sense, WO99/25376 describes a method of eliciting a T cell immune response against an antigen in a mammal which comprises administering to said mammal an auxotrophic attenuated strain of Listeria which expresses the antigen. Said auxotrophic attenuated strain is described therein as having a mutation in at least one gene whose protein product is essential for growth of the Listeria. In particular, the invention describes an auxotrophic attenuated strain for the synthesis of D-alanine which further comprises DNA encoding a heterologous antigen, wherein the heterologous antigen is preferably an HIV-1 antigen.
In WO99/25376, the results are presented as showing that the auxotrophic strain of Listeria provides protection against challenge by L. monocytogenes in BALB/c mice making this strain alledgely suitable for use in a vaccine composition for protection against an infection caused by this organism. However, the experimental examples provided therein merely establish that attenuated auxotrophic D-alanine mutants of L. monocytogenes elicit a CTL (host cytotoxic T cell) response. An antibody-mediated immune response (humoral immunity) is not considered therein nor are results provided in this sense. Moreover, the protection effect of this mutant is therein determined by measuring the bacterial counts in the spleen of infected mice after challenge with the wild type Listeria. In this sense, the effectiveness of a vaccine against acute and lethal bacterial infections (especially those causing sepsis) can only be asessed if survival assays are conducted. In addition, mutant Listeria injected without D-alanine are described therein as providing little protection. In contrast, when D-alanine was supplemented in the initial inoculum of the mutant organism to achieve the same protection as the wild type strain (at the time of initial immunization), this had the effect of reducing the lethal dose of the mutant about 10 fold, a serious limitation for the safety of this mutant if the lost of attenuation is considered. Therefore, the results provided in WO99/25376 for the D-alanine mutants therein described, fail to demonstrate the uselfulness of these mutant strains as vaccine candidates against extracellular bacterial pathogens and acute systemic infections. Moreover, the lack of cross-protection data with these mutants does not assure the effectiveness of a vaccine composed of D-alanine auxotrophs to generate a broadly protective immune response against other L. monocytogenes strains, much less its usefulness for generating vaccine candidates in other bacterial species.
In addition, in the detailed description of WO99/25376, the inventors make the following suggestion: “Additional potential useful targets for the generation of additional include the genes involved in the synthesis of the cell wall component D-glutamic acid. To generate D-glutamic acid auxotrophic mutants, it is necessary to inactivate the dat gene, which is involved in the conversion of D-glu+pyr to alpha-ketoglutarate+D-ala and the reverse reaction. It is also necessary to inactivate the glutamate racemase gene, dga”. However, one of ordinary skill in the art will know that there is no reasonable expectation of success in light of the information presented therein that D-glutamic acid auxotrophic strains of Listeria can presumably confer a satisfactory level of attenuation to avoid unacceptable pathological effects and elicit an adequate immune response capable of conferring a lasting protection in the host against the disease (protective immunity). As discussed above, each gene-targeted strategy should be evaluated case by case. Moreover, the glutamate racemase enzyme has moonlinghtening functions that can affect celular viability if its coding genes are manipulated. In this sense, the use of glutamate racemase as a target to generate D-glutamate auxotrophic vaccine strains to confer protection against bacterial infections cannot be extrapolated from the previous invention, because there is no sustained evidence presented, and it is not obvious that such a strain can be immunogenic.
The latter statement, namely that it is not obvious that such D-glutamate auxotrophic vaccine strain can be immunogenic, is further sustained by the fact that there are no studies nor inventions demonstrating the ability of D-glutamate auxotrophic micro-organisms to be useful as live vaccines for conferring protection against bacterial-caused diseases in the current state of vaccine development. In this sense, and despite the considerable level of attenuation of a D-glutamic acid auxotrophic mutant demonstrated in M. P. Cabral et al, “Blockade of glutamate racemisation during cell-wall formation prevents biofilm and proliferation of Acinetobacter baumannii in vivo”, Abstract of the 23rd ESCMID congress (European Society of Clinical Microbiology and Infectious Diseases) held in Berlin from the 27th to the 30th of April, 2013 (the only reference showing a relation between D-glutamate auxotrophy and in vivo loss of virulence), it is noted that one of the main obstacles to the development of vaccines is the difficulty in achieving a satisfactory level of attenuation without severely compromising immunogenicity (protection). So, correlation between attenuation and protection need to be invariably tested for each gene-targeted modification strategy in order to develop an effective vaccine against bacterial infections. In this sense, the above mentioned document (M. P. Cabral et al, “Blockade of glutamate racemisation during cell-wall formation prevents biofilm and proliferation of Acinetobacter baumannii in vivo”, Abstract of the 23rd ESCMID congress (European Society of Clinical Microbiology and Infectious Diseases) held in Berlin from the 27th to the 30th of April, 2013), even thought it describes the attenuation of an Acinetobacter baumannii strain characterized by the in-frame deletions of glutamate racemase genes, fails to provide any data showing the protective efficacy of such a strain against A. baumannii infections. Providing data showing the protective efficacy of an attenuated strain is crucial to determine the usefulness of such a strain as a vaccine as demonstrated in the following prior art documents.
In M. K. Hondalus et al, “Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis”, Infection and Immunity 68 (2000) 2888-2898, a leucine auxotroph of M. tuberculosis was created by allelic exchange so that the mutant was unable to replicate in macrophages (proving that the bacteria was attenuated). In fact, FIG. 5 of this document shows how mice infected with the leucine auxotroph of M. tuberculosis had a 100% survival rate 22 weeks post-infection (establishing that leucine auxotroph was indeed attenuated). However, the leucine auxotrophic mutant was shown to be less effective than the live BCG vaccine in reducing organ burdens and tissue pathology of BALB/c mice challenged intravenously. This document illustrates that it is not enough to have an attenuated strain to have a vaccine and that immunogenicity is a key issue.
Furthermore, in M. S. Jr Pavelka et al, “Vaccine efficacy of a lysisne auxotroph of Mycobacterium tuberculosis”, Infection and Immunity 71 (2003) 4190-4192, it was demonstrated that a single intravenous immunization of mice with the M. tuberculosis mutant (a lysine auxotroph of M. tuberculosis) did not generate a significant protective response to the subsequent aerosol and that a single immunization with the auxotroph was insufficient for reducing the bacterial burden in the lungs and spleens relative to a single immunization with BCG. Only the triple immunized mice survived as long as the control mice immunized with a single dose of BCG. Consequently, again it can be concluded that immunogenicity is a key and separate issue from attenuation.
Moreover, prior art reference Ann-Mari Svennerholm et al, “Vaccines against enterotoxigenic Escherichia coli”, Expert review of vaccines 7 (2008) 795-804, describes genetically attenuated ETEC (enterotoxigenic Escherichia coli) strains as live oral vectors and characterized as safe. However when evaluating these same strains for protection, neither the attack rate for diarrhea nor the total stool volume was significantly diminished in vaccines versus placebo recipients.
Lastly, H. K. Kim et al, “Identifying protective antigens of Staphylococcus aureus, a pathogen that suppresses host immune responses”, FASEB J. 25 (2011) 3605-3612, describes whether three attenuated mutants derived from the Newman strain by transposon insertional mutagenesis can elicit protective immunity in mice. These mutants were constructed in order to block the expression of exoproteins, surface proteins as well as the processing of surface proteins, namely saeR (S. aureus exoprotein), mgrA (multiple gene regulator A) and srtA (sortase A). However, mutants lacking saeR or mgrA, despite being attenuated in mice, did not to confer protective immunity to subsequent S. aureus infection.
Consequently, even if a reduced virulence (good level of attenuation) for a particular derivative strain is achieved, its protective capacity in the host must be experimentally assessed to be able to conclude its usefulness as a live vaccine.